Wilms tumor gene-1 (“WT1”) is a gene that was identified as a causative gene of pediatric Wilms tumor by Call et al. in 1990 (Non-patent Literature 1). Thereafter, it has been indicated that WT1 mRNA is expressed at a high rate in not only pediatric Wilms tumor but also solid cancer cells, such as solid cancer cell lines, e.g., gastric cancer cell lines, colon cancer cell lines, lung cancer cell lines, and breast cancer cell lines (Non-patent Literature 2). The WT1 gene is now considered to be a cancer-related gene associated not only with pediatric Wilms tumor but also many cancers.
Call et al. report the expression of WT1 mRNA in K562 cells and CCRF-CEM cells, which are both leukemia cell lines (see Non-patent Literature 1). Miwa et al. report that WT1 mRNA was expressed in 15 of 22 cases of acute myeloid leukemia (“AML”) in northern blot analysis (Non-patent Literature 3). Further, Inoue et al. report that the expression of WT1 mRNA was observed in 100% (45/45) or cases at the first medical examination for AML (Non-patent Literature 4). In addition, it is reported that the expression level of WT1 mRNA at diagnosis is associated with prognosis (Non-patent Literature 5), that even if the expression of WT1 mRNA returns to normal levels with treatment, it increases again when recurrence occurs (Non-patent Literature 6), and that the expression level of WT1 mRNA at recurrence is higher than at diagnosis (Non-patent Literature 7).
The WT1 gene has been sold as an extracorporeal diagnostic pharmaceutical product that is useful as a new marker for monitoring minimal residual disease (or “MRD”) in the treatment for AML because of the fact that the WT1 gene appears with high frequency as a single gene in patients with AML and the expression of the WT1 gene increases again at recurrence after return to normal levels with treatment.
A hitherto-known method for measuring human WT1 mRNA is a competitive quantification method using β-actin as a standard (Patent Literature 1). However, this method requires not only measuring WT1 mRNA and β-actin mRNA separately, but also performing extension reactions after a reverse transcription reaction is done, i.e., two-step RT-PCR, thus requiring a lot of time.
As another method for measuring human WT1 mRNA, Patent Literature 2 discloses one-step RT-PCR for WT1 mRNA. However, this method requires the expression level of a housekeeping gene used for correcting the expression level of the WT1 gene to be separately measured, and thus is complicated.